Iron deficiency is one of the most common human nutritional disorders in the world today (Yip, R. (1994) J. Nutr. 124: 1479S-1490S). Indeed, iron is an essential nutrient for virtually all organisms because it plays a critical role in important biochemical processes such as respiration and photosynthesis. Although abundant in nature, iron is often available in limited amounts because the oxidized form, Fe(III), is extremely insoluble at neutral or basic pH. This fact is of particular importance to agriculture because approximately one-third of the world""s soils are classified as iron-deficient (Yi, Y. et al. (1994) Plant Physiol. 104: 815-820). Many xe2x80x9ciron-efficientxe2x80x9d plant varieties have iron uptake strategies (designated strategy I or strategy II) that, not surprisingly, are directed at solubilizing iron (Rxc3x6mheld, V. (1987) Physiol. Plant. 70: 231-234). Strategy II plants, which include all of the grasses, release Fe(III) compounds called xe2x80x9cphytosiderophoresxe2x80x9d into the surrounding soil that bind iron and are then taken up into the roots. Most other iron-efficient plants use strategy I and respond to iron deprivation by inducing the activity of membrane-bound Fe(III) chelate reductases that reduce Fe(III) to the more soluble Fe(II) form. The Fe(II) product is then taken up into the roots by an Fe(II) specific transport system that is also induced by iron-limiting growth conditions. Furthermore, the roots or strategy I plants release more protons when iron-deficient, lowering the rhizosphere pH and thereby increasing the solubility of Fe(III). Thus, it would be desirable to take advantage of this understanding of iron-uptake strategies to produce plants which have increased iron-uptake capabilities.
Furthermore, another metal, zinc, is an integral cofactor of many proteins and is indispensable to their catalytic activity and/or structural stability (Vallee and Auld (1990) Biochemistry 9:5647-5659). Moreover, zinc is a ubiquitous component of enzymes involved in transcription and of accessory transcription factors, the zinc finger proteins, that regulate gene expression (Rhodes and Klug (1993) Sci. Am. 268(2):56-65). Because of the many roles this metal plays in cellular biochemistry, zinc is an essential nutrient for all organisms. Despite this importance, very little is known about the molecular mechanisms cells use to obtain zinc. No transporter genes involved in zinc uptake (i.e. influx transporters) have been isolated from any organism. Recently, genes have been identified whose products are responsible for detoxifying intracellular zinc by transporting the metal from the cytoplasm to the cell exterior or into intracellular compartments (i.e. efflux transporters). These genes include the closely related eukaryotic genes, COT1, ZRC1, and Znt-1 (Conklin et al. (1992) Mol. Cell Biol. 12:3678-3688; Kamizono et al. (1989) Mol. Gen. Genet. 219:161-167; Palmiter and Findley (1995) EMBO J. 14:639-649). While important for zinc detoxification, these genes do not appear to play a role in zinc uptake.
In addition, metal ion pollution is perhaps one of the most difficult environmental problems facing the industrial world today. Unlike the organic and even halogenated organic pollutants, which can be degraded in the soil, metals are essentially nonmutable. The electrolytic, in situ immobilization and chemical leaching technologies for cleaning polluted sites are all very expensive, particularly in light of how vast some of these sites are. With the exception of approaches like vitrification, most in situ metal ion remediation schemes require some mechanism for increased mobilization of the metal ion. This raises the possibility of further endangering local wildlife or adjacent ecosystems not already affected. Thus, a need still exists for better methods for removing toxic pollutants from the soil.
Accordingly, an object of the invention is to generate transgenic plants in which expression of an MRT polypeptide is altered such that metal-uptake is increased.
Another object of the invention to provide methods for removing toxic pollutants, such as heavy metals, from the environment.
Yet another object of the invention is to provide methods for improving human or animal nutrition, e.g., for treating metal-deficiency, e.g., iron-deficiency or zinc-deficiency.
This invention is based, at least in part, on the discovery of a family of polypeptides, designated herein as metal-regulated transporter, MRT, polypeptides, which share several structural/functional properties, at least one of which is related to metal transport. Structurally, the MRT polypeptides include, for example, at least one transmembrane binding domain which has at least about 40%, more preferably at least about 50%, 55%, 60%, 70%, 80% or 90% amino acid sequence identity with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14 and/or at least one histidine rich domain. Functionally, the MRT polypeptides are capable of, for example, transporting metals, e.g., Fe, e.g., Fe(II), Cd, Co, Mn, Pb, Hg and/or Zn.
Preferred MRT polypeptides have an overall amino acid sequence identity of at least about 40%, preferably at least about 42%, 45%, 47%, 50%, more preferably at least about 55%, 60%, 70%, 80%, 90%, or 95% with an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14; it has eight transmembrane domains; it has four histidine rich domains; or it can be isolated from the Arabidopsis family of plants.
Accordingly, this invention pertains to isolated nucleic acid molecules encoding an MRT polypeptide. Such nucleic acid molecules (e.g., cDNAs) have a nucleotide sequence encoding an MRT polypeptide (e.g., an A. thaliana IRT1 polypeptide, an A. thaliana IRT2 polypeptide, an A. thaliana ZIP1 polypeptide, an A. thaliana ZIP2 polypeptide, or an A. thaliana ZIP3 polypeptide) or biologically active portions or fragments thereof, such as a polypeptide having an MRT bioactivity. In a preferred embodiment, the isolated nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13, or a portion or fragment thereof. Preferred regions of these nucteotide sequences are the coding regions. Other preferred nucleic acid molecules are those which have at least about 45%, preferably at least about 48%, more preferably at least about 50%, and most preferably at least about 55%, 60%, 70%, 80%, 90%, 95%, 97% or 98% or more nucleotide sequence identity over the entire sequence with a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13, or a portion or fragment thereof. Nucleic acid molecules which hybridize under stringent conditions to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13, e.g., nucleic acid molecules which hybridize to at least 6 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13, are also within the scope of the invention. Such portions or fragments include nucleotide sequences which encode, for example, polypeptide domains having an MRT bioactivity. Examples of portions or fragments of nucleic acid molecules which encode such domains include portions or fragments of nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13 which encode one or more of the following: at least one transmembrane domain which has at least about 40%, more preferably at least about 50%, 55%, 60%, 70%, 80% or 90% amino acid sequence identity with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14 or at least one histidine rich domain. Nucleic acid molecules of the present invention which further comprise a label are also within the scope of the invention. Complements of the nucleic acid molecules of the present invention are also specifically contemplated.
In another embodiment, the nucleic acid molecules of the invention encode a polypeptide having an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14, or a portion or fragment thereof having a biological activity, e.g., an MRT bioactivity. Nucleic acid molecules encoding a polypeptide having at least about 40%, preferably at least about 42%, 45%, 47%, 50%, more preferably at least about 52%, and most preferably at least about 55%, 60%, 70%, 80%, 90%, 95%, 97% or 98% amino acid sequence identity over the entire sequence with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14, or a portion or fragment thereof having a biological activity, e.g., an MRT bioactivity, are also within the scope of the invention.
Another aspect of the invention pertains to nucleic acid molecules which encode polypeptides which are fragments of at least about 20 amino acid residues in length, more preferably at least about 30 amino acid residues in length or more, of an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14. Other aspects of the invention pertain to nucleic acid molecules which encode polypeptides which are fragments of at least about 20 amino acid residues in length, more preferably at least about 30 amino acid residues in length which have at least about 40%, more preferably at least about 42%, 45%, 47%, 50%, and most preferably at least about 55%, 60%, 70%, 80%, 90% or more (e.g., 95%, 97-98%) amino acid sequence identity over the entire sequence with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14, or a portion or fragment thereof having a biological activity, e.g., an MRT bioactivity. Portions or fragments of the polypeptides encoded by the nucleic acids of the invention include polypeptide regions which comprise, for example, various structural and/or functional domains of MRT family members. Such domains include portions or fragments of nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:13 which encode one or more of the following: at least one transmembrane domain which has at least about 40%, more preferably at least about 50%, 55%, 60%, 70%, 80% or 90% amino acid sequence identity with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14, or at least one histidine rich domain. Nucleic acid molecules which are antisense to the nucleic acid molecules described herein are also within the scope of the invention.
Another aspect of the invention pertains to vectors, e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention and host cells into which such recombinant expression vectors have been introduced. In one embodiment, such a host cell is used to produce an MRT polypeptide by culturing the host cell in a suitable medium. An MRT polypeptide protein can be then isolated from the medium or the host cell.
Still another aspect of the invention pertains to isolated MRT polypeptides (e.g., isolated A. thaliana IRT1 polypeptides) and active fragments thereof, such as peptides having an activity of an MRT polypeptide (e.g., at least one biological activity of an IRT1 polypeptide as described herein). The invention also provides an isolated or purified preparation of an MRT polypeptide. In preferred embodiments, an MRT polypeptide comprises an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14. In other embodiments, the isolated MRT polypeptide comprises an amino acid sequence having at least about 40%, more preferably at least about 42%, 45%, 47%, 50%, and most preferably at least about 55%, 60%, 70%, 80%, 90% (e.g., 95%, 97%-98%) or more amino acid sequence identity over the entire sequence with an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14, and, preferably has an activity of an MRT polypeptide (e.g., at least one biological activity of MRT). Preferred MRT polypeptides include, for example, at least one transmembrane binding domain which has at least about 40%, more preferably at least about 50%, 55%, 60%, 70%, 80% or 90% amino acid sequence identity with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14, and/or at least one histidine rich domain. Preferred MRT polypeptides are capable of, for example, transporting metals, e.g., Fe, e.g., Fe(II), Cd, Co, Mn, Pb, Hg and/or Zn.
Fragments of the MRT polypeptides of the invention can include portions or fragments of the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14, which are at least about 20 amino acid residues, at least about 30, or at least about 40 or more amino acid residues in length. The MRT polypeptide portions or fragments described herein can have an MRT bioactivity, e.g., one or more, in any combination, of the MRT biological activities described herein. Portions or fragments of the polypeptides of the invention can include polypeptide regions which comprise, for example, various structural and/or functional domains. Such domains include portions or fragments of amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14, which include at least one of the following: a transmembrane domain which has at least about 40%, more preferably at least about 50%, 55%, 60%, 70%, 80% or 90% amino acid sequence identity with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14, or a histidine rich domain. Preferred amino acid sequences of each of these domains are described herein. The peptide fragments can be modified to alter MRT bioactivity, e.g., impart a non-wild type activity on MRT polypeptides, or to impart desired characteristics thereon, e.g., increased solubility, enhanced therapeutic or prophylactic efficacy, or stability. Such modified peptides are considered functional equivalents of peptides having an activity of MRT as defined herein. A modified peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition. In another embodiment, a component which imparts a desired characteristic on a peptide can be linked to the peptide to form a modified peptide.
The invention also provides for an MRT fusion polypeptide comprising an MRT polypeptide and a second polypeptide portion having an amino acid sequence from a protein unrelated to an amino acid sequence which has at least about 40% or more amino acid sequence identity with an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14.
The invention also provides transgenic plants in which the expression of an MRT polypeptide is altered, as well as seeds and cells derived from such plants. For example, the invention includes a method for evaluating the effect of the expression or misexpression of an MRT gene on a parameter related to metal transport. The method includes providing a transgenic plant having an MRT transgene, or which otherwise misexpresses an MRT gene, contacting the transgenic plant with an agent, and evaluating the effect of the transgene or misexpression of the MRT gene on the parameter related to metal transport (e.g., by comparing the value of the parameter for a transgenic plant with the value for a control, e.g., a wild-type plant).
In addition, the transgenic plant, e.g., rice, beans, peas and maize, in which expression of an MRT polypeptide is altered can be incorporated into a pharmaceutical composition which includes the transgenic plant, or a portion thereof, and a pharmaceutically acceptable carrier. Such compositions can be used as human or animal nutritional supplements to provide, for example, iron to a subject with iron-deficiency or zinc to a subject with zinc-deficiency. Antibodies, e.g., monoclonal or polyclonal antibodies, which bind to an epitope of or are specifically reactive with an MRT polypeptide or fragment thereof are also specifically contemplated in the present invention.
Methods for identifying an agent which inhibits or activates/stimulates an MRT polypeptide are also within the scope of the invention. These methods include contacting a first polypeptide comprising a naturally occurring ligand of MRT, with a second polypeptide comprising an MRT polypeptide and an agent to be tested and then determining binding of the second polypeptide to the first polypeptide. Inhibition of binding of the first polypeptide to the second polypeptide indicates that the agent is an inhibitor of an MRT polypeptide while activation/stimulation of binding of the first polypeptide to the second polypeptide indicates that the agent is an activator/stimulator or an MRT polypeptide.
In another aspect, the invention features a method for evaluating a candidate compound for the ability to interact with an MRT polypeptide. This method includes contacting the compound with the MRT polypeptide and evaluating the ability of the compound to interact with the MRT polypeptide. This method can be performed in vitro or in vivo.
The MRT polypeptides of the invention can be used to modulate metal concentrations in vitro or in vivo. In one aspect, the invention provides a method for modulating metal concentration in a biological sample containing the metal. This method includes providing a transgenic plant in which expression of an MRT polypeptide is altered and contacting the transgenic plant with the biological sample such that the metal concentration in the biological sample is modulated.
The invention further provides methods for removing a pollutant from soil. These methods include contacting a transgenic plant in which expression of an MRT polypeptide is altered with the soil such that the pollutant is removed from the soil. In a preferred embodiment, the pollutant is a metal, e.g., a metal selected from the group consisting of Pb, As, Co, Cu, Zn, Cd and/or Hg.
Additional methods of the invention include methods for treating a disorder associated with metal-deficiency, e.g., iron-deficiency or zinc-deficiency, in a subject. These methods include administering to a subject a therapeutically effective amount of a composition comprising a transgenic plant, or a portion thereof, in which expression of an MRT polypeptide is altered. In a preferred embodiment, the composition is administered in combination with a pharmaceutically acceptable carrier. In other preferred embodiments, the MRT polypeptide in the transgenic plant is overexpressed. In yet other preferred embodiments, the disorder associated with iron-deficiency is anemia.
Still additional methods of the invention include methods for promoting plant growth and/or survival. These methods include introducing into a plant a nucleic acid encoding an MRT polypeptide.